4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors demonstrating good in vivo efficacy

Article abstract of DOI:10.1016/j.bmcl.2012.06.065

The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent and selective IKK2 inhibitor showing good efficac

Full text of DOI:10.1016/j.bmcl.2012.06.065

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5222–5226
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
4-Phenyl-7-azaindoles as potent, selective and bioavailable IKK2 inhibitors
demonstrating good in vivo efficacy
John Liddle a, , Paul Bamborough a, Michael D. Barker a, Sebastien Campos a, Chun-Wa Chung a,
⇑
Rick P. C. Cousins a, Paul Faulder a, Michelle L. Heathcote a, Heather Hobbs a, Duncan S. Holmes a,
Chris Ioannou a, Cesar Ramirez-Molina a, Mary A. Morse a, Ruth Osborn b, Jeremy J. Payne a,
John M. Pritchard a, William L. Rumsey b, Daniel T. Tape a, Giorgia Vicentini a, Caroline Whitworth a,
Rick A. Williamson a
a GlaxoSmithKline R&D, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
b GlaxoSmithKline Inc., 709 Swedeland Road, King of Prussia, PA 19406, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The lead optimization of a series of potent azaindole IKK2 inhibitors is described. Optimization of the
human whole blood activity and selectivity over IKK1 in parallel led to the discovery of 16, a potent
and selective IKK2 inhibitor showing good efficacy in a rat model of neutrophil activation.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 11 May 2012
Revised 18 June 2012
Accepted 20 June 2012
Available online 26 June 2012
Keywords:
IKK2
Kinase
Azaindole
The I
pathway by which the NF-
vated in response to pro-inflammatory stimuli such as lipopolysac-
charide (LPS) and tumor necrosis factor (TNF B signaling
).1–4 NF-
j
B kinases (IKKs) are essential components of the signaling
inhibition of IKK2 activity offers a promising mechanism for inter-
vention in a range of inflammatory diseases.11
j
B p50/RelA transcription factor is acti-
a
j
results in the expression of numerous genes involved in innate and
adaptive immune responses and the pathway is also implicated in
chronic inflammatory disorders including rheumatoid arthritis
(RA), chronic obstructive pulmonary disorder (COPD) and asthma.
Table 1
Profile of lead azaindole 1
CH3
O
N
The I
j
B kinase complex contains two catalytic subunits, IKK1 and
B essential modulator (NEMO).5
S
OH
O
IKK2, and a regulatory subunit NF-
j
Although IKK1 and IKK2 are highly homologous serine–threonine
protein kinases (61% of amino acids are identical in their aligned ki-
nase domains whereas the ATP binding sites differ by only one ami-
no acid) and contain similar structural domains, studies in
knockout mice and derived mouse embryonic fibroblasts suggest
that IKK2 is the predominant kinase involved in IkB phosphoryla-
CH3
N
H
N
tion and hence NF-j
B activation.6,7 IKK1 deficient mice present an
unexpected phenotype including skeletal and skin abnormalities.8
Furthermore, IKK1 appears to be involved in the control of cell cycle
through direct phosphorylation of Aurora A in the nucleus and may
contribute to inflammatory resolution.9,10 Therefore, selective
Assay
pIC50
IKK2
IKK1
A549 Cell
LPS/TNF hWB
HSA binding
7.4
5.5
6.1
5.5
91%
pIC50 values are reported as mean of P2 experiments.
Assay details are given in Ref. 13. IKK1 and IKK2 activity at 1
HSA is human serum albumin binding.
l
M ATP.
⇑
Corresponding author.
E-mail address: john.2.liddle@gsk.com (J. Liddle).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.06.065

J. Liddle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5222–5226
5223
Table 2
In vitro profile of analogues 2–9
H
N
O
S
N
O
O
S
O
R
N
H
Compd
R
IKK2 pIC50
IKK1 pIC50
LPS/TNF
6.4
a hWB pIC50
2
CH3
7.7
7.9
3
8.3
8.2
6.4
4
5
6
8.2
8.4
8.1
8.2
8.2
7.6
6.6
6.6
6.2
Figure 1. X-ray structure of 4 bound to JNK1, viewed from the same position as the
model of IKK2 shown in Figure 1 of Ref. 12.
Table 3
7
8
7.7
7.8
7.0
7.5
5.9
6.2
N
N
In vitro profile of analogues 2, 10–12
H
N
O
NH
S
O
O
S
X
Y
O
N
N
9
7.8
7.3
5.9
Z
pIC50 values are reported as mean of P2 experiments.
Assay details are given in Ref. 13.
CH3
N
H
N
Compd
X
Y
Z
IKK2 pIC50
IKK1 pIC50
LPS/TNF hWB pIC50
We recently reported our early lead optimisation efforts and
preliminary SAR around a series of ATP competitive azaindole
IKK2 inhibitors.12 Compound 1 (Table 1) was disclosed as a potent
and highly selective inhibitor with good cellular activity in a mech-
2
CH
N
CH
CH
CH
CH
N
CH
CH
CH
N
7.7
7.9
7.1
6.1
7.9
8.1
7.1
6.8
6.4
6.4
6.0
5.5
10
11
12
N
anistic cell assay (inhibition of TNFa induced NF-jB reporter acti-
vation in A549 cells).13 Herein, we report our extended studies on
this template and conclude with the discovery of a potent, selec-
tive, IKK2 inhibitor with good oral efficacy in a rat in vivo model
of lung neutrophil activation.
pIC50 values are reported as mean of P2 experiments.
Assay details are given in Ref. 13.
ally tolerant of a range of substituents and hence this area was uti-
lised in an attempt to increase IKK2 potency and introduce
selectivity over IKK1. 2-Ethyl, isopropyl, cyclopropyl and t-butyl
analogues 3–6 were more potent than 2 at IKK2, and maintained
good activity in the whole blood assay, but offered no advantage
in terms of selectivity over IKK1 (Table 2). The increase in IKK2 po-
tency observed with the 2-alkyl substituent is likely due to a
hydrophobic interaction within the outer lipophilic pocket of the
ATP binding site.
Interestingly, the basic dimethylamino compound 7 was toler-
ated at IKK2 but showed modest selectivity over IKK1. The 4-piper-
idine analogue 8 was also potent at both IKK2 and IKK1 with
micromolar activity in the whole blood assay. Several analogues
containing large 2-substituents were prepared to probe the outer
regions of the IKK2/1 catalytic sites in an attempt to exploit any
subtle conformational differences and gain selectivity. Although
potency at IKK2 was maintained along with good whole blood
activity, analogues failed to show an acceptable selectivity window
over IKK1. For example, triazole 9 has excellent IKK2 activity but
insufficient selectivity over IKK1.
Although 1 showed good IKK2 enzyme inhibition in the bio-
chemical assay, approximately 100 fold reduction in activity was
observed in the whole blood cellular assay (inhibition of LPS stim-
ulated TNFa
in human whole blood).13 The drop-off in activity ob-
served in the whole blood assay reflects a number of factors
including plasma protein binding, cellular permeability and com-
petition with endogenous concentrations of ATP. Given that 1 rep-
resents an attractive ligand efficient lead (ligand efficiency
index = 4.2, logD 2.8, MW 345), the aim of the programme was
to increase the whole blood activity and introduce adequate phar-
macokinetics across preclinical species whilst maintaining the
excellent kinase selectivity.12
Several sulphonamide analogues were prepared illustrating
that excellent IKK2 enzyme inhibition can be achieved with this
series of 4-phenyl-7-azaindoles as previously described.12 How-
ever, few analogues had satisfactory whole blood activity
(pIC50 >5.5). One of the more potent compounds discovered early
in the campaign, sulphone 2 (Table 2), was more potent than 1
in the IKK2 biochemical assay and this increase in activity was mir-
rored with a superior whole blood activity (pIC50 6.4).
In the absence of an IKK2 protein X-ray structure, we utilised a
Although 2 had excellent whole blood activity, the compound
lacked selectivity over IKK1. The azaindole 2-position was gener-
homology model to dock potential binding modes.14 As previously

5224
J. Liddle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5222–5226
Table 4
pair of interactions with the hinge (Met111, the JNK1 equivalent
In vitro profile of analogues 13–17
of Cys99 in IKK2). The sulfonamide adopts the same orientation
in the IKK2 model as in the JNK1 crystal structure, interacting
with the conserved lysine (Lys55 in JNK1). The cyclic sulfone
H
N
O
S
OH
O
makes no specific interactions with JNK1, although one of the
0
SO2 oxygens does lie within 3.0 ÅA of the sidechain oxygen of
Ser155. In IKK2, the Ser residue is replaced by the glutamic acid
Glu149, therefore no direct hydrogen-bonding interaction is
likely. The lack of any obvious favourable IKK2 interactions is
consistent with the relatively flat SAR observed with the sulph-
onamide moiety.
R
N
H
N
Introduction of heteroatoms into the central aryl ring did not im-
prove IKK2 potency or selectivity (Table 3). The pyridyl compound
10 maintained good activities at both IKK2 and IKK1. The southern
pyridyl isomer 11 lost activity at both IKK2 and IKK1 and was essen-
tially equipotent at both isoforms. The pyrimidyl compound 12 was
a weak IKK2 inhibitor (pIC50 6.1) and showed a marginal preference
for IKK1. These observations probably result from a combination of
factors. The first is that polar nitrogen atoms are disfavoured at the
southern position because of the proximity to the hydrophobic
sidechains of Val29, Met96, and Ile165 in IKK2 (equivalent to
Val40, Met108 and Leu168 in JNK1, Fig. 1). The second is that nitro-
gen atoms adjacent to the bi-aryl bond would favour a more planar
torsion angle than 30°, which was observed in the JNK1 X-ray
complex.
Compd
R
IKK2 pIC50
IKK1 pIC50
LPS/TNF hWB pIC50
13
14
CH3
CF3
7.7
7.8
6.5
6.7
5.6
5.6
15
16
17
8.1
8.1
7.7
6.4
6.7
6.1
5.8
5.9
6.0
pIC50 values are reported as mean of P2 experiments.
Assay details are given in Ref. 13.
Although the initial hydroxyethyl sulphonamides 1 and 13 had
moderate whole blood activity, they were the most selective over
the IKK1 isoform. The reasons for this are unclear, since all of the
residues in the ATP site of IKK2 and IKK1 are identical, with the
exception of one position located next to the hinge (Gln100 in
IKK2, Ser99 in IKK1). However, this points away from the binding
site. Any selectivity must be due to conformational effects trans-
mitted to the binding site from the outer region. The SAR observed
at the azaindole 2-position of the 2-hydroxyethyl sulphonamides
(Table 4) was similar to that observed with the sulphones in Table
reported, the model suggests that the 7-azaindole binds at the
hinge region of the ATP site with a pair of hydrogen-bonds to the
backbone of Cys99, with the 2-position pointing towards the outer
edge of the site. The sulfonamide substituent was predicted to lie
close to the conserved lysine (Lys44).12
To gain confidence in this model, crystallisation was at-
tempted with several surrogate kinases. A structure was solved
with 4 bound to JNK1 (JNK1 pIC50 of 6.0).15 In this structure
(Fig. 1), the azaindole does indeed form the hydrogen-bonding
Table 5
In vitro profile and rat PK profile of compounds 15, 16 and 18–21
H
N
O
S
R
O
1
R
N
H
N
Compd
R
R1
IKK1 pIC50
IKK2 pIC50
LPS/TNF hWB pIC50
cLogP
Rat PK summary
IV Cl mL/min/kg IV t1/2 (h)
F%
OH
15
8.1
6.4
5.8
2.9
39
0.45
18
OH
OH
16
18
8.1
8.2
6.7
6.5
5.9
6.0
2.4
3.1
13
14
0.6
0.7
49
100
OH
19
20
21
CH3
8.0
8.1
7.7
6.7
6.6
6.5
6.0
6.3
5.9
2.7
2.7
2.8
70
15
7
0.6
0.9
3.2
32
29
83
OH
OH
CF3
pIC50 values are reported as mean of P2 experiments. Assay details are given in Ref. 13.
IV administration (n = 2) 1 mg/kg. Oral administration (n = 1) 1 mg/Kg. Cl is the in vivo clearance from IV administration.

J. Liddle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5222–5226
5225
Table 6
Compounds 15 and 16 had favourable physicochemical proper-
ties, good selectivity and whole blood potency and hence were pro-
filed in a rat pharmacokinetic study alongside several close
analogues listed in Table 5. Interestingly, the 2-substituent gener-
ally had a significant effect on the rat in vivo clearance and hence
on oral bioavailability. The 2-methyl and isopropyl analogues gen-
erally had higher clearance than the 2-cyclopropyl and 2-CF3 ana-
logues; the isopropyl analogue 15 had moderate clearance (ꢀ50%
liver blood flow) and poor bioavailability of 18%. The correspond-
ing cyclopropyl compound 16, which is of similar lipophilicity to
15, had lower rat in vivo clearance and good oral bioavailability
of 49%. Both 15 and 16 had a low volume of distribution (0.8 and
0.6 L/Kg, respectively) leading to a short half life of less than one
hour. The high in vivo clearance consistently observed with the
2-methyl and 2-isopropyl analogues may reflect a greater propen-
sity for oxidation on the pseudo-benzylic CH and hence phase I
metabolism.
In vitro profile and physicochemical properties of 16
Parameter
Result
IKK2/IKK1 (pIC50
A549 cell (pIC50
Whole blood LPS-TNF
MW/clog P
CYP450 (IC50
Intrinsic clearance (hepatocytes)
hERG (pIC50
Plasma protein binding (%)
Aqueous solubility
)
8.1/6.7
6.7
)
a
(pIC50
)
5.9 (human), 5.2 (rat)
357/2.4
7 lM (2C9), >10 lM (3A4, 2D6, 1A2)
<1.0 (human), <1.0 (rat) (mL/min/g)
<4.3
97.6 (rat), 93.4 (human)
64 lM
)
)
Assay details are given in Ref. 13.
2. The trifluoromethyl analogue 14 was essentially equipotent to
13 in the biochemical and whole blood assay. Isopropyl and cyclo-
propyl derivatives 15 and 16 showed an increase in IKK2 potency
with a modest improvement in whole blood activity. The t-butyl
analogue 17 was equipotent to 13 but again marginally more ac-
tive in the whole blood assay.
Methyl substitution was tolerated on the hydroxyethyl chain
but had marginal affect on the biochemical and cellular activity
of the series. Good rat pharmacokinetic profiles were obtained
Figure 2. Dose dependent inhibition of neutrophil infiltration in rat lungs with compound 16.
Cl
N
Me
b
a
c
N
NH
N
H
N
H
N
N
O
N
H
+
-
O
O
25
24
23
22
H
O
O
H
N
N
O
O
S
N
OH
H
N
O
O
S
B
O
O
OH
Cl
S
S
OH
d
e
f
Br
O
O
Br
N
H
26
27
28
16
Scheme 1. Reagents and conditions: (a) N-Methoxy-N-methylcyclopropanecarboxamide, nBuLi, THF, ꢁ5 °C, 1 h then 5 M HCl, 50 °C, 18 h, 86%. (b) m-Chloroperoxybenzoic
acid, DME, 0–20 °C, 7 h, 58%. (c) Methanesulphonyl chloride, DMF, 60 °C, 2 h, 50%. (d) Ethanolamine, triethylamine, DCM, 0 °C, 3 h then 20 °C, 16 h, 81%. (e) 4,4,40,40,5,5,50,50-
octamethyl-2-20-bi-1,3,2-dioxaborolane, palladium acetate, potassium acetate, DMF, 55 °C, 4 h, 77%. (f) 25, potassium phosphate, 20-(dimethylamino)-2-biphenyl-
palladium(II) chloride dinorbonylphosphine, dioxane, water, 100 °C, 1 h, 60%.

5226
J. Liddle et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5222–5226
with several analogues. For example, the lipophilic (clog P 3.1)
gem-dimethyl analogue 18 maintained low rat in vivo clearance
(14 mL/min/kg), typical of the majority of 2-cyclopropyl analogues,
and a high bioavailability of approximately 100%. The correspond-
ing 2-methyl analogue 19 had high in vivo clearance (70 mL/min/
kg), and a moderate bioavailability of 32%. The homochiral ana-
logues 20 and 21 also had attractive in vitro profiles. However,
20 had poor oral bioavailability of 29% in the rat. 21 had excellent
bioavailability of 83% in the rat when dosed at 1 mg/kg but suffered
from poor aqueous solubility compared to other analogues. Such
low solubility is likely to preclude achieving sufficiently high sys-
temic concentrations required for in vivo toxicity evaluation.
Compound 16 had the preferred overall profile including low
molecular weight (357), optimal log P (2.4), good solubility and a
free fraction of approximately 7% in human blood (Table 6). Fur-
References and notes
1. Barnes, P. J.; Karin, M. N. Eng. J. Med. 1997, 336, 1066.
2. Li, Q.; Verma, I. M. Nat. Rev. Immunol. 2002, 2, 725.
3. Karin, M.; Yamamoto, Y.; Wang, Q. M. Nat. Rev. Drug Disc. 2004, 3, 17.
4. Hayden, M. S.; Ghosh, S. Gen. Dev. 2004, 18, 2195.
5. Yamamoto, Y.; Gaynor, R. B. Trends Biochem. Sci. 2004, 29, 72.
6. Li, J.; Peet, G. W.; Pullen, S. S. J. Biol. Chem. 1998, 273, 30736.
7. Hu, Y.; Baud, V.; Delhase, M. Science 1999, 284, 316.
8. Strnad, J.; Burke, J. R. Trends Pharm. Sci. 2007, 28, 142.
9. Prajapati, S.; Tu, Z.; Yamamoto, Y.; Gaynor, R. B. Cell Cycle 2006, 5, 2371.
10. Lawrence, T.; Bebien, M.; Liu, G. Y.; Nizet, V.; Karin, M. Nature 2005, 434, 1138.
11. Bamborough, P.; Callahan, J. F.; Christopher, J. A.; Kerns, J. K.; Liddle, J.; Miller,
D. D.; Morse, M. A.; Rumsey, W. L.; Williamson, R. Curr. Top. Med. Chem. 2009, 9,
623.
12. Liddle, J.; Bamborough, P.; Barker, M. D.; Campos, S.; Cousins, R. P. C.; Cutler, G.
J.; Hobbs, H.; Holmes, D. S.; Ioannou, C.; Mellor, G. W.; Morse, M. A.; Payne, J. J.;
Pritchard, J. M.; Smith, K. J.; Tape, D. T.; Whitworth, C.; Williamson, R. A. Bioorg.
Med. Chem. Lett. 2009, 19, 2504.
13. The IKK2 enzyme assay, NF-jB cellular mechanistic assay, human whole blood
thermore, 16 had an acceptable P450 profile (2C9 IC50 = 7
lM,
assay and azaindole syntheses are described in WO2008/034860.
14. A homology model of IKK2 was built as previously described: Christopher, J. A.;
Avitabile, B. G.; Bamborough, P.; Champigny, A. C.; Cutler, G. J.; Dyos, S. L.;
Grace, K. G.; Kerns, J. K.; Kitson, J. D.; Mellor, G. W.; Morey, J. V.; Morse, M. A.;
O’Malley, C. F.; Patel, C. B.; Probst, N.; Rumsey, W.; Smith, C. A.; Wilson, M. J.
Bioorg. Med. Chem. Lett. 2007, 17, 3972.
other isoforms IC50 >10 M) with no evidence of time dependent
l
inhibition at CYP3A4 and 2D6. Importantly, 16 had low intrinsic
clearance when incubated with human hepatocytes.
Compound 16 also had excellent kinase selectivity. When
screened against over 60 in-house kinases (including ROCK, JNK1,
Aurora A and B), only IKK1 inhibition was within 30 fold of IKK2
inhibition (fold selectivity based on comparative IC50 values).
The in vivo efficacy of 16 was evaluated in a rat model of
neutrophil activation, a standard animal model of inflammation.
15. E. coli expressed GST-tagged truncated human Jnk1alpha (1-364) was purified
to homogeneity as detailed in Bioorg. Med. Chem. Lett. 2009, 19, 360. The
protein–ligand complex, concentrated to between ꢀ8–11 mg/ml in 50 mM Tris
pH 7.6, 150 mM NaCl, 10 mM DTT, was crystallized against a well solution
consisting of 0.05 M ammonium sulfate, 0.05 bis-Tris pH 6.5, 30% v/v
Pentaerythritol (15/4 EO/OH). Crystals were improved by use of seeding.
Data from a single frozen crystal was collected at the European Synchrotron
Radiation Facility, Grenoble and processed using MOSFLM and scaled using
SCALA to give a 1.90 Å dataset. The P212121 cell (a = 50.438 Å, b = 72.747 Å,
c = 110.155 Å, a = b = g = 90°) has a single molecule in the ASU. After rigid body
refinement using a previously determined in house structure, model building
was performed using Coot and refined using REFMAC. There was clear
difference density for the ligand in the ATP binding site and the binding
mode could be unambiguously determined. The final model (R/Rfree = 18.8/
21.9%) has been deposited in the protein databank under the accession codes
4awi.pdb.
16 was dosed orally at 10, 30 and 100 lmol/kg to male rats
30 min prior to the rats being exposed to aeorosolised LPS.16 16
was found to inhibit neutrophil lung infiltration in a dose depen-
dent manner giving approximately 50% reduction with a corre-
sponding plasma concentration of approximately 2
lM (Fig. 2).
At 30 mol/kg po (equivalent to approximately 11 mg/kg), there
l
was a significant reduction in neutrophil infiltration as compared
to those animals receiving the vehicle.17 This data is consistent
16. Compound 16 was dissolved in 2% DMSO with 0.5% methylcellulose for a final
with the potency value obtained using the rat LPS/TNFa cellular as-
say (pIC50 = 5.2)
concentration of 10
assist in solubilisation. Dilutions were made with 0.5% methylcellulose to
obtain concentrations of 3 and 1 mol/ml. Male Lewis rats were dosed orally at
a volume of 10 ml/kg 30 min prior to LPS aerosol. Final doses were 10, 30 and
100 mol/kg. Each group, including vehicle, was n = 4. Thirty minutes
following the compound dose, the rats were exposed to aerosolized 0.1 mg/
ml LPS solution from E. coli, serotype 026:B6 (Sigma, St. Louis) at a rate of 4.5 L/
min for 20 min. At 4 h-post LPS exposure the rats were euthanized by Fatal Plus
i.p. (100 mg/kg). Bronchoalveolar lavage (BAL) was performed through a 14
gauge blunt needle inserted into the exposed trachea in five, 5 ml washes of
Dulbecco’s phosphate buffered saline (DPBS) to collect a total of 20–23 ml of
BAL fluid. Blood was taken via cardiac puncture for DMPK analysis. BAL fluid
was centrifuged at 1000–1500 rpm for 10 min. Supernatant was aspirated and
lmol/kg (3.57 mg/ml). 200 ll 1 N NaOH was added to
l
The compounds described in this paper were prepared by previ-
ously reported chemistry.13 Analogues were generally prepared via
a divergent route involving a final Suzuki reaction coupling the
azaindole core to the phenylsulphonamide moiety. The synthetic
route to 16 is shown in Scheme 1. An initial lithiation of a Boc-
protected 2-amino-3-methyl pyridine and condensation with
N-methoxy-N-methylcyclopropanecarboxamide furnished the
cyclopropyl-azaindole 23. Oxidation of the azaindole followed by
addition of methane sulphonyl chloride yielded the 4-chloroazain-
dole 25. Routine chemistry furnished the borane ester 28 and sub-
sequent Suzuki reaction gave 16 in 60% yield.
In summary, we have presented the lead optimisation of a ser-
ies of potent and selective azaindole IKK2 inhibitors. The medicinal
chemistry strategy was focussed on maintaining the excellent
physicochemical properties of the lead 1 whilst optimising the
whole blood activity and rodent pharmacokinetics; this led to
the discovery of 16 which had an excellent physicochemical and
in vitro profile. Furthermore, 16 had good rat pharmacokinetics
and demonstrated in vivo efficacy in a rat model of neutrophil
activation.
l
discarded. Cell pellet was resuspended in 5 ml PBS. An aliquot of 70–100 ll
was centrifuged on a Shandon cytospin at 300 rpm for 5 min. Slides were
stained with Diff-Quick (a Wright-Giemsa stain) for differential cell counts.
Total cell counts were performed with
calculated as inhibition of each dose as compared to vehicle-treated
animals and and ED50 was calculated of 28.2 mol/kg (10.1 mg/kg). Plasma
a haemocytometer. Data were
%
l
concentrations at 4.5 h post-compound administration were determined.
17. Statistical analysis using Anova followed by Post Hoc Dunnett’s test confirmed
that the 30 lmol/kg dose led to significant inhibition compared to control.
The human biological samples were sourced ethically and their research use
was in accord with the terms of the informed consents.
All animal studies were ethically reviewed and carried out in accordance with
animals (scientific procedures) Act 1986 and the GSK Policy on the Care,
Welfare and Treatment of Animals.